For the mensuration of imaged specimen structures, the dimensions of the specimen region in question are associated with a selected image region and are indicated or displayed. In the previous size calibration operation, for example in the case of a digital image, the length and height of a pixel are associated with the length and height of the corresponding specimen region. For measurement of a specimen structure, the latter is marked, the number of associated pixels is identified, and the dimension in the specimen plane is determined on the basis of the calibration value. This method is used in particular for the mensuration of microscopic structures; here, for example in the case of a microscope, for each optical magnification the specimen size corresponding to one pixel is stored e.g. in the form of a lookup table. For example, the pixel size (e.g. in the horizontal direction) is 1 μm in the specimen space at a 10× optical magnification, 0.5 μm at a 20× magnification, 0.25 μm at a 40× magnification, and so forth. If the pixel size in the horizontal direction differs from that in the vertical direction, the table entries need to be enhanced accordingly. For calibration of an image, the corresponding list entry is then selected in accordance with the selected image acquisition conditions (usually the particular optical magnification), in order to obtain the relevant pixel size, i.e. the current calibration value.
A number of methods are known for obtaining the appropriate calibration values in optical instruments having differing selectable magnifications.
In a first method, hereinafter called the list method, a micrometer specimen, for example a grating having a specific grating spacing, is introduced into the optical instrument, the optical magnification of the instrument being known. The micrometer specimen is imaged, and the corresponding image is displayed, for example, on a computer monitor. A distance (for example in the horizontal direction) is then marked on the image of the micrometer specimen, and the number of pixels within that distance is ascertained. From the ratio between the actual length of the distance (which is known for the micrometer specimen in question) and the number of pixels, the calibration value is then obtained for the selected or specified optical magnification of the instrument.
Unlike in the mathematical method explained below, in the list method the associated calibration value is then calculated for each possible setting of the optical instrument (typically, for each possible optical magnification), and stored in a list or lookup table. In contrast to this, in the mathematical method the calibration value stored for a specific optical magnification is also used for images acquired using a different optical magnification, by modifying the stored calibration value on the basis of the ratio between magnifications (stored reference magnification ratio and magnification currently in use).
Downstream from the optical instrument there is often a digital camera which further processes the image from the optical instrument, records it, and/or forwards it to a computer on whose monitor it is displayed to a user. Digital cameras can, however, usually be operated in different reproduction modes. There exists, for example, a “DC 100” digital camera of the Applicant having a normal reproduction mode (765×581 pixels) and an interpolated reproduction mode (1146×871 pixels), or e.g. the “DC 350 F” digital camera having a normal mode of 1300×1030 pixels and a binning mode of 650×515 pixels. The aforementioned size calibration methods are applicable in error-free fashion only if a digital camera is operated in exactly the reproduction mode in which the calibration took place. If operation is switched over into another mode, a size calibration must be performed again, since because of the change in number of pixels per image region, the previous calibration value would furnish incorrect results for mensuration.
It is usual, therefore, to generate and save a list having calibration values for every possible camera reproduction mode. Since there can be a large number of reproduction modes (e.g. Normal, Binning, Interpolated, Multi-Shot, etc.), the present method results in long lists, especially when each possible magnification of the optical instrument additionally needs to be taken into consideration. It has been found that generation of the lists (lookup tables), and access times to the table entries upon mensuration of a specimen, are time-intensive and moreover carry the risk that an incorrect camera mode will be selected from the list.